Methods for Diagnosing and Treating Kidney Cancer

ABSTRACT

Methods, reagents and kits for diagnosing and treating kidney cancer are disclosed. An immunoassay for detecting kidney cancer is based on the relative change of the CELSR1 protein in urine or blood compared with normal tissue. An immunohistochemical assay for detecting kidney cancer is based on the relative absence of labeled antibody binding to cancerous tissue, compared with normal tissue.

This application is a divisional application of U.S. application Ser.No. 11/363,596, filed 27 Feb. 2008, the entire contents of which areexpressly incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This application relates generally to methods for diagnosing andtreating kidney cancer.

REFERENCES

The following references are cited below in support of the background ofthe invention or methods employed in practicing the invention.

-   1. Wu, Q. & Maniatis, T. Large exons encoding multiple ectodomains    are a characteristic feature of protocadherin genes. Proc Natl Acad    Sci USA 97, 3124-9 (2000).-   2. Hadjantonakis, A. K. et al. Celsr1, a neural-specific gene    encoding an unusual seven-pass transmembrane receptor, maps to mouse    chromosome 15 and human chromosome 22qter. Genomics 45, 97-104    (1997).-   3. Hadjantonakis, A. K., Formstone, C. J. & Little, P. F. mCelsr1 is    an evolutionarily conserved seven-pass transmembrane receptor and is    expressed during mouse embryonic development. Mech Dev 78, 91-5    (1998).-   4. Tissir, F., De-Backer, O., Goffinet, A. M. & Lambert de    Rouvroit, C. Developmental expression profiles of Celsr (Flamingo)    genes in the mouse. Mech Dev 112, 157-60 (2002).-   5. Nusse, R., van Ooyen, A., Cox, D., Fung, Y. K. & Varmus, H. Mode    of proviral activation of a putative mammary oncogene (int-1) on    mouse chromosome 15. Nature 307, 131-6 (1984).-   6. Nusse, R. & Varmus, H. E. Many tumors induced by the mouse    mammary tumor virus contain a provirus integrated in the same region    of the host genome. Cell 31, 99-109 (1982).-   7. Sorensen, A. B., Duch, M., Amtoft, H. W., Jorgensen, P. &    Pedersen, F. S. Sequence tags of provirus integration sites in DNAs    of tumors induced by the murine retrovirus SL3-3. J Virol 70,    4063-70 (1996).-   8. Lund, A. H. et al. Genome-wide retroviral insertional tagging of    genes involved in cancer in Cdkn2a-deficient mice. Nat Genet 32,    160-5 (2002).-   9. Mikkers, H. et al. High-throughput retroviral tagging to identify    components of specific signaling pathways in cancer. Nat Genet 32,    153-9 (2002).-   10. Collier, L. S., Carlson, C. M., Ravimohan, S., Dupuy, A. J. &    Largaespada, D. A. Cancer gene discovery in solid tumours using    transposon-based somatic mutagenesis in the mouse. Nature 436, 272-6    (2005).-   11. Dupuy, A. J., Akagi, K., Largaespada, D. A., Copeland, N. G. &    Jenkins, N. A. Mammalian mutagenesis using a highly mobile somatic    Sleeping Beauty transposon system. Nature 436, 221-6 (2005).-   12. Suzuki, T. et al. New genes involved in cancer identified by    retroviral tagging Nat Genet 32, 166-74 (2002).-   13. Li, J. et al. Leukaemia disease genes: large-scale cloning and    pathway predictions. Nat Genet 23, 348-53 (1999).-   14. Lovmand, J. et al. B-Cell lymphoma induction by akv murine    leukemia viruses harboring one or both copies of the tandem repeat    in the U3 enhancer. J Virol 72, 5745-56 (1998).-   15. van Lohuizen, M. et al. Identification of cooperating oncogenes    in E mu-myc transgenic mice by provirus tagging. Cell 65, 737-52    (1991).

BACKGROUND OF THE INVENTION

Cancer is caused by genetic aberrations, i.e., mutations. In mutantcells the normal balance between the factors that promote and restraingrowth is disrupted, and as a result, these mutant cells proliferatecontinuously—the hallmark of tumor cells. Mutations can arisespontaneously or by external factors such as chemical mutagens,radiation, or viral integration, which inserts extra-genomic DNA thatmay or may not contain an oncogene. A cellular gene can be modified bypoint mutation, insertion and frame shift (including truncation),(functional) deletion (including silencing), or translocation, whichsometimes can result in gene fusion. In this way proto-oncogenes becomeoncogenes, which promote proliferation, and tumor suppressor genesbecome inactivated, also inducing tumor growth. Any combination of theabove-mentioned changes in DNA can contribute to tumor formation. Thereare two ways by which mutations result in transformation: the expressionlevel of the genes is changed, or their function is altered. Theconsequences of these changes may or may not be held in check by theimmune system (immune surveillance).

Heretofore, there has been no demonstrated link between changes inCELSR1 levels and kidney cancer. Such a link could have a number ofimportant diagnostic and therapeutic applications. In accordance withthe present invention, it has now been discovered that (i) CELSR1 levelschange, e.g., drop significantly in kidney cancer cells, and (ii) thischange can be measured the blood-fluid and urine sample of patients.

SUMMARY OF THE INVENTION

The invention includes, in one aspect, a histological method forexamining human kidney tissue for the presence and extent of kidneycancer. This method involves the steps of staining the kidney tissuewith an antibody specific against a selected domain or epitope of CELSR1and labeled with a detectable marker, to attach the marker to thesurface of tissue cells having surface bound CELSR1 protein with thatepitope or domain, and determining, based on a reduced distribution andextent of detectable marker with respect to the distribution and extentof marker in normal kidney cells, the presence and extent of kidneycancer in the tissue.

In various embodiments, the antibody may be specific against an epitopecontained within SEQ ID NO:1, (i) a cadherin domain, such as containedwithin a sequence identified by SEQ ID NOS: 2-9, (ii) an EGF_CA domain,such as contained within a sequence identified by SEQ ID NOS: 10, 11,13, 15, and 16, (iii) a LamG domain, such as contained within a sequenceidentified by SEQ ID NOS: 12 and 14, (iv) an EGF_Lam domain such ascontained within the sequence identified by SEQ ID NO: 17, and (v) otherdomains, such as contained within sequences identified by SEQ ID MOS:18-20.

In another aspect, the invention includes an antibody that is specificagainst an epitope contained with amino acid residues 90-150 of humanCELSR1, and defined by SEQ ID NO:1. The antibody may be labeled with adetectable marker suitable for immuno-histochemical detection ofcancerous kidney or neuronal tissue, based on the relative absence ofhistochemical staining of the tissue compared with normal kidney orneuronal issue, respectively.

Also disclosed is a method for identifying genetic mutations associatedwith an increased risk of kidney cancer. The method involves (a)extracting genomic DNA from cells from cancerous kidney tissue fromhuman patients, (b) for the DNA extracted from cells from each tissue,comparing the sequence of the DNA in a selected region of the CELSR1gene and its 5′-UTR and 3′-UTR, with a homologous region of DNA fromcells from normal, wildtype human kidney tissue, and (c), by thiscomparing, identifying one or more mutations in said regions associatedwith an increased risk kidney cancer. The selected region of the genefrom at least one of (i) a plurality of exons 1 to 35 of the CELSR1 onchromosome interval 22q13.3, including adjacent splice site acceptor anddonor sequences of the exons, (ii) a 5′ UTR region within 10 kB or lessof exon 1 of the gene, and (iii) a 3′-UTR region within 10 kB or less ofexon 35. One exemplary region is a 5′ UTR region within 10 kB or less ofexon 1 of the CELSR1 gene.

The method may be in constructing a gene chip designed for geneticscreening for risk of kidney cancer, For each mutation identified instep (c), a gene fragment capable binding selectively to genomic DNAfragments carrying that mutation, but not to corresponding wildtype DNAfragments is produced, and the different-sequence fragments are attachedat known positions on a gene-chip substrate.

In still another aspect, there is provided a method of screening forkidney cancer in a human subject, or staging treatment of kidney cancerin a subject, by reacting a body-fluid sample from the subject with anantibody specific against a selected domain or epitope of CELSR1, anddetermining from the presence and/or amount of immunoassay product,whether the subject has a reduced level of CELSR1 protein lacking thespecific domain or epitope, when compared with a normal range of CELSR1in human samples, as an indicator of kidney cancer. The body-fluidsample may be urine, and the assayed level of CELSR1 indicative ofkidney cancer may be a level less than about 0.1 ng/ml.

The method may be carried out by applying the body fluid to asolid-phase immunoassay device, the level of CELSR1 in the sample may beindicated qualitatively by a calorimetric or fluorometric indicator, andthe determining step may include comparing the indicator with a knownstandard.

In a related aspect, the invention includes a diagnostic device for usein screening for kidney cancer in a human subject, or staging treatmentof kidney cancer in a subject. The device comprises (a) a structure forreceiving a body-fluid sample from the subject, (b) an antibody specificagainst a selected domain or epitope of CELSR1, and associated with thestructure and capable of reacting with body-fluid received in saidstructure, to produce, in combination with other reagents associatedwith the structure, a detectable reaction indicative of the level ofCELSR1 in the sample, and (c) a known-standard indicator against whichthe level of detectable reaction produced can be assessed as an elevatedlevel associated with kidney cancer.

The structure in the device may include a porous pad having theanti-CELSR1 binding protein embedded therein, for reaction with thefluid sample when the sample is applied to the pad, the detectablereaction may be indicated by a colorimetric or fluorimetric indicator,and the known standard indicator may include an indicia that representsa level of CELSR1 corresponding to that associated with kidney cancer.

The device may be employed in a kit which includes a spectrophotometricdetector for generating a signal related to the level of CELSR1produced, a microprocessor for comparing the signal with a know-standardsignal value associated with kidney cancer, and a display for displayingan output of the microprocessor.

Also provided by the invention is a method of treating kidney cancer ina subject by the steps of (a) reacting a body-fluid sample from thesubject with an antibody specific against a selected domain or epitopeof CELSR1, (b) determining from the presence and/or amount ofimmunoassay product, whether the subject has a reduced level of CELSR1protein lacking the specific domain or epitope, when compared with anormal range of CELSR1 in human samples, as an indicator of kidneycancer, and (c) if the subject has such a reduced CELSR1 level,administering a therapeutically effective amount of a CELSR1 bindingagent effective, when bound to the surface of kidney cancer cells, toinhibit growth or viability of the cells. One exemplary antibody in themethod is a human or humanized anti-CELSR1 antibody specific against anepitope contained within SEQ ID NO:1.

These and other aspects, objects, advantages, and features of theinvention will become apparent to those persons skilled in the art uponreading the details of the invention as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the genomic organization of the human CELSR1 gene (a.k.a.flamingo 2) and a schematic representation of the protein it encodes.

FIG. 2 shows the genomic organization of the mouse Celsr1 locus, asviewed by a customized screen print of the UCSC genome web site browser(March 2005 version of the mm6 gene assembly). Top, base position onchromosome 15. Vertical green bars represent the retroviral insertionsinto the locus in 6 independent tumors.

FIGS. 3A-3B show an example of immunohistochemical stains of a humankidney tumor (renal cell carcinoma) (FIG. 3A), and matched normal kidneytissue (FIG. 3B) from the same patient. The polyclonal rabbit antibodyused reacts to an epitope that is found between amino acid residues 90and 150, which is encoded by exon 1.

FIGS. 4A and 4B show a solid-phase diagnostic device for determiningCELSR1 levels in a human patient, at initial (4A) and final stages (4B)of the assay; and

FIG. 5 shows a portion of a gene chip useful for diagnosing geneticpredisposition to kidney cancer, constructed in accordance with thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION A. Definitions

The following terms have the definitions given below, unless otherwiseindicated in the specification.

“Screening” for kidney cancer, in accordance with the present invention,means testing individuals for a level of CELSR1 that is indicative ofkidney cancer or an elevated risk of kidney cancer.

“Staging” treatment of kidney cancer, in accordance with the presentinvention, involves determining the stage of kidney cancer in anindividual, based on the level of CELSR1 detected, and tailoring thetreatment to that stage. There are four recognized stages of kidneycancer, which are defined by the degree of localization of cancer cells.In addition, kidney cancer may be defined as early stage at which thecancer is responsive to a number of hormonal-based therapies, and alater, more serious androgen-independent stage.

“A reduced level of CELSR1 protein” may include, as an indicator ofcancer, a reduced level of wildtype CELSR1 protein or a reduced level ofCELSR1 protein having a specific epitope or domain. That is, either theabsence of any CELSR1 protein or the presence of a defective CELSR1protein may be indicative of cancer, e.g., kidney cancer.

B. CELSR1 Protein and Expression

The cadherin EGF LAG seven-pass G-type receptor-1 (CELSR1) protein¹⁻³,encoded by the CELSR1 gene (FIG. 1), is a member of the flamingosubfamily, which, in turn, is part of the cadherin superfamily. Theflamingo subfamily consists of nonclassic-type cadherins, which do notinteract with catenins. The flamingo cadherins are located at the plasmamembrane and have at least eight cadherin domains, at least fivecalcium-binding epidermal growth factor-like (EGF_CA) domains, twolaminin A G-type (LamG) domains, a laminin-type EGF-like (EGF_Lam)domain, and at least three receptor domains identified as (1) the HormRdomain (a domain present in hormone receptors), (2) a GPS domain(G-protein-coupled receptor proteolytic site domain), and a 7tm domain(7-transmembrane receptor domain from the secretin family). The aminoacids sequences identified with these domains are given in the SequenceListing below. It is believed that CELSR1 proteins are receptorsmediating contact-mediated communication. The cadherin domains may actas homophilic binding regions, and the EGF-like domains may be involvedin cell adhesion and receptor-ligand interactions.

The human unprocessed CELSR1 protein precursor has 3014 amino acidresidues (AA) and a calculated molecular weight (MW) of 329486 Da; themouse precursor has 3034 AA and a calculated MW of 330481 Da. The humangene contains 35 exons; all 9 protocadherin ectodomain repeats of thehuman protein are encoded by the large first exon¹; in the mouseortholog, this exon is split into exons 1 and 2. Part of the human exon1, without all the other exons, is also transcribed in lung epithelialcells (EST CA944897), with a predicted protein of 188 AA.

The CELSR1 protein is expressed in the brain, where it is localizedprincipally in the ependymal cell layer, choroid plexus and the areapostrema; and in the spinal cord and in the eye. It is a developmentallyregulated, neural-specific gene which plays an unspecified role in earlyembryogenesis. First detected at developmental stage E6 in the mouse, itis predominantly expressed in the developing central nervous system(CNS), the emerging dorsal root ganglia and cranial ganglia. In the CNS,expression is uniform along the rostrocaudal axis. During gastrulation,it is expressed in the vicinity of the primitive streak, and becomespredominant in that area at late gastrulation. At E10, it is detected inventricular zones (VZ), but not in marginal zones (MZ), and weakly inother structures. Between E12 and E15, it is highly expressed in the VZin all brain areas, but not in differentiated neuronal fields. In thenewborn and postnatal stages, expression remains restricted to the VZ,but it is also weakly expressed in fetal lungs, kidney and epithelia 4.

C. Screening for CELSR1-Related Cancers in Mice

Cancer genes (oncogenes and tumor suppressor genes) were defined in ahigh throughput manner by using proviral tagging. Although viruses havenot yet been implicated as a major cause of cancers in humans, researchusing tumor viruses has led to the discovery of many oncogenes andprotooncogenes. In proviral tagging, mice are infected with a retrovirusthat does not contain an oncogene (e.g., murine leukemia virus, MLV ormurine mammary tumor virus, MMTV)⁵⁻⁹. Recently, the host range of thisapproach has been broadened by the use of a transposon^(10, 11).

During retroviral infection, the virus integrates into the cellulargenome and inserts its DNA near or within genes, which leads to variousoutcomes: (i) The insertion site is too far away from a protooncogeneand thus does not activate it. In this case, there will be no selectionfor that cell. (ii) The provirus inserts within 200 kb of aprotooncogene, but not within the gene (type 1). Here, either the viralpromoter or the viral enhancer increases the expression level of theprotooncogene. (iii) The provirus inserts within a gene, destroying oraltering its function (type 2). There will be no selection for a cellthat contains either type 1 or type 2 insertion events in a gene that isnot a protooncogene or tumor suppressor gene. If integration results inthe formation of a tumor, genes adjacent to the integration site can beidentified, and classified as either protooncogenes or tumor suppressorgenes. This method has been used to identify protooncogenes as well asto confirm already known protooncogenes discovered by virtue of theirhomology to viral oncogenes^(5, 6, 18, 9, 12-15). A tumor suppressor maybe scored if a retrovirus lands within a gene and truncates or destroysit. In these cases, the suppressor may be haplo-insufficient, oralternatively, the mutation on the other allele is providedspontaneously by the mouse. The integration event may also lead to morecomplex consequences, such as a dominant negative effect of thetruncated gene product or the transcription of anti-sense or microRNA.

In a screen with T lymphotrophic virus SL3-3, six independent tumorswere recovered that contained provirat integrations in the mouse CELSR1locus: five tumors contained integrations within intron 2 (whichcorresponds to human intron 1) while one tumor contained an integrationwithin intron 6 (which corresponds to human intron 5) (FIG. 2). In thefirst five tumors, all 9 protocadherin ectodomain repeats are predictedto be separated from the rest of the protein. In tumor 6, theprotocadherin domains plus part of the EGF-like motif are predicted tobe separated from the rest of the protein. The results demonstrate thatdisruption of the cadherin and/or EGF-like domains in CELSR1 protein cantrigger a tumor, and that therefore, tumors may be associated withCELSR1 proteins lacking a critical domain or epitope or with absence ofthe CELSR1 protein itself.

D. Expression of CELSR1 in Human Tumors and in Normal Tissue

The mutations that were identified as causal in mouse tumor formationdisrupted the gene after exon 2. In humans, exon 1 encompasses thesequences of both exons 1 and 2 of the mouse. In addition to the signalpeptide, human exon 1 encodes the 9 cadherin domains of CELSR1. As thecadherin domains extend to AA 1197, a rabbit polyclonal antibody to anepitope that is included between AA 90 to 150 was used for the studieson oncogenic mutations that caused the (functional) loss of theectodomains. Indeed, this domain was found to be missing in renal cellcarcinomas (FIG. 3), whereas the normal counterpart of these tumor cellsstrongly expressed CELSR1 (FIG. 3). Clearly, the absence of part or allof the ectodomains is a marker for these tumors.

More generally, the invention provides a histological method forexamining human kidney tissue for the presence and extent of cancer. Inthe method, kidney tissue is stained with a labeled antibody specificagainst a selected domain or epitope of CELSR1, e.g.,fluorescence-labeled antibody (see Section E below), to attach themarker to the surface of tissue cells. The presence and extent of kidneycancer in the tissue is then determined based on a reduced distributionand extent of detectable marker with respect to the distribution andextent of marker in normal kidney cells.

E. Preparation of Anti-CELSR1 Antibody

This section describes production of anti-CELSR1 antibodies useful fordiagnostic and therapeutic purposes, as described further in thesections below. The anti-CELSR1 antibody used in the present inventioncan be obtained by any a variety of conventional methods as amonoclonal, polyclonal, or recombinant antibody. One preferred antibody,particularly for diagnostic use, is a mouse monoclonal antibody,prepared according to well-known hybridoma methodology. Briefly, humanCELSR1 may be first obtained, for example, by expressing the CELSR1(MXR7) gene as disclosed by Lage, H. et al (Gene 188 (1997), 151-156).The purified CELSR1 protein acts as an immunogen. Alternatively, apartial peptide of CELSR1 can be used as a sensitization antigen. Inparticular, for generating antibodies specific against a selectedepitope or domain of CELSR1, a peptide defining that domain or epitopemay be used as the immunogen. These peptides can be defined by thesequences given in the Sequence Listing below. For example, to generatean antibody specific against an epitope contained in SEQ ID NO:1, thepeptide defined by this sequence is employed as the immunogen.

Anti-CELSR1 antibodies useful in diagnostic applications may be labeledwith a variety of detectable labels, including detectable reporters,such as enzymes for enzyme-linked immunosorbent assays (ELISA),detectable particles, such as gold particles and reporter-carryingliposomes, colorimetric or fluorescent reporters, labels such as quantumdot nanocrystal particles, radiolabels, and labels such as a biotinlabel by which secondary detectable labels, such as a reporter-labeledstreptavidin label can be attached. In some assay formats, an unlabeledanti-CELSR1 antibody, for example, a mouse IgG antibody, is detected byreaction with a labeled antibody, e.g., a labeled anti-mouse IgGantibody.

For therapeutic uses, human monoclonal antibodies having bindingactivity to CELSR1, (see Japanese Patent Publication (Kokoku) No.1-59878 B (1989)) can be produced by sensitizing in vitro humanlymphocytes with CELSR1, and causing the sensitized lymphocytes to fusewith the human-derived myeloma cells having a permanent divisionpotential. Alternatively, CELSR1 as an antigen can be administered to atransgenic animal having all the repertories of a human antibody gene toobtain anti-CELSR1 antibody-producing cells, and then human antibodiesfor CELSR1 may be obtained from the immortalized anti-CELSR1antibody-producing cells (see International Patent Publication Nos. WO94/25585, WO 93/12227, WO 92/03918 and WO 94/02602).

In still other methods, human or humanized antibodies specific againstCELSR1 antigen can be prepared by recombinant technique, such as havebeen reported (see, for example, U.S. Pat. Nos. 6,090,382 and6,258,562).

F. Diagnostic Methods and Reagents

In one aspect, the invention includes a method of screening for kidneycancer in a human subject, or staging treatment of kidney cancer in asubject. This is done, in accordance with the invention, by reacting abody-fluid sample from the subject with an antibody specific against aselected domain or epitope of CELSR1, and determining from the presenceand/or amount of immunoassay product, whether the subject has a reducedlevel of CELSR1 protein lacking the specific domain or epitope, whencompared with a normal range of CELSR1 in human samples, as an indicatorof kidney cancer.

Preferred body-fluid samples are blood and urine. Where urine isassayed, the assayed level of CELSR1 indicative or kidney cancer istypically in the range less than about 0.1 ng/ml sample fluid.

The assay may be carried out by any of a variety of assay methods usedfor detecting body-fluid antigens, including ELISA techniques,homogeneous assays, for example, involving fluorescence quenching, and avariety of solid-phase sandwich assays in which the CELSR1 antigen iscaptured on by an anti-CELSR1 antibody carried on a solid support, andthe immobilized antigen-antibody complex is labeled with a secondanti-CELSR1 antibody, e.g., a second antibody carrying a calorimetric orgold-particle reporter.

FIGS. 4A and 4B illustrate a solid-phase assay strip constructed inaccordance with an embodiment of the invention, suitable for carryingout a sandwich immunoassay of the type just mentioned, and shown ininitial and final assay states, respectively. The strip, indicatedgenerally at 10, includes a porous support or pad 12 having asample-application zone 14 in an upstream region of the support and asample-detection zone 16 in a downstream region. The sample-applicationzone includes a detectable anti-CELSR1 antibody reagent, e.g.,anti-CELSR1 antibodies labeled with gold particles, and carried in thezone in an unbound, i.e., non-immobilized form. This reagent isindicated by solid circles, such as at 18. Anti-CELSR1 antibodies, whichmay be the same or different from those in the labeled antibody reagent,are immobilized to the solid support within the detection zone, and areindicated by the “Y” shapes, such as at 20.

Also shown is a reference zone 22 which is located adjacent thedetection zone and has one or more colored or shaded regionscorresponding to different assay levels of CELSR1 in a body-fluidsample. In the embodiment shown, zone 22 includes three regions 22 a, 22b, and 22 c, corresponding to an assayed level of CELSR1 (a) below thatassociated with kidney cancer, (b) corresponding to a lower thresholdlevel associated with kidney cancer, and (c) a level that issubstantially higher, e.g., 2-3 times, higher than the threshold layerin region 22 b, respectively. These three regions provide a knownstandard indicator against which the level of detectable reactionproduced can be assessed as a level associated with kidney cancer.Together, the assay strip and reference zone constitute an assay devicefor use in screening for kidney cancer in a human subject, or forstaging treatment of kidney in a human subject.

In operation, a known volume of a body-fluid sample to be tested isadded to the sample-application zone of the strip, where it diffusesinto the zone, allowing the antibody reagent to react with CELSR1antigen in the sample to form an antigen-antibody complex. This complexand unbound antibody reagent then migrate downstream by capillaritytoward the detection zone, where the antigen-antibody complex iscaptured by the immobilize antibody and the unbound reagent is carriedto the end of the support, as indicated at 24. As can be appreciated,the higher the concentration of antigen in the body fluid, the higherthe density of captured reagent in the detection zone and the greaterthe color or intensity in this zone. This color or intensity produced inthe detection zone is compared with the standards in the reference zoneto determine a qualitative level of CELSR1 associated with the presenceor absence of kidney cancer. If a sub-threshold level or threshold levelof CELSR1 is observed in the assay, the subject can be classified in ahigher-probability category for the presence of cancer, and the subjectmay be recommended for additional testing and/or more frequent testing.

In another embodiment, the assay device includes an assay strip likethat described above, but where the known-reference indicator isprovided by a strip-reader instrument reader having (i) a reader slotfor receiving the assay strip, (ii) a light source and an opticaldetection, e.g., a spectrophotometric detector, for detecting anassay-related optical condition at the detection zone of the assaystrip, (iii) an electronics or processor unit which records andprocesses a signal from the optical detector, and converts the signal toan assayed level of CELSR1, and (iv) a user display screen or window.The instrument may report the actual CELSR1 body-fluid sample detected,allowing the operator to compare the displayed value with known standardindicator levels provided with the assay strip or instrument, to assesswhether the subject has an reduced CELSR1 level associated with kidneycancer, or to assess the possible stage of the cancer, for purposes oftreatment design. Alternatively, the instrument itself may containstored known-standard indicator levels which can be compared internallywith an assayed level to generate an output that indicates whether anreduced CELSR1 level associated with kidney cancer has been detected, orto indicate the stage of the cancer.

G. Identifying Genetic Mutation Associated with Kidney Cancer

In another aspect, the invention provides a method for identifyingmutations associated with increased risk of kidney cancer in a humansubject. In practicing the method, genomic DNA is extracted from humanpatients having kidney cancer, preferably including patients from men orwomen representing different racial and age groups. The DNA sequencesthat are examined, in particular, are (i) one or more of exons 1 to 35of the CELSR1 on chromosome interval 22q13.3, including adjacent splicesite acceptor and donor sequences of the exons, (ii) a 5′ UTR regionwithin 10 kB or less of exon 1 of the gene, and (iii) a 3′-UTR regionwithin 10 kB or less of exon 35.

Mutations at one or more sites along the region are identified bycomparing each of the sequences with sequences from the same regionderived from normal (wildtype) kidney tissue. Preferably sequences froma number of wildtype individuals are determined to ensure a truewildtype sequence. For each extracted DNA, the patient and wildtypesequences are compared to identify mutations in the patient sequences,and thus mutations that are likely associated with increased risk ofkidney cancer.

Once a large number of these mutations are identified, e.g., at least50-200 or more, they may be used in constructing a genetic screeningdevice, e.g., a gene chip, useful for screening individuals for geneticpredisposition to kidney cancer. In one embodiment, the device includesa gene chip, such as shown at 30 in FIG. 5, having an array of regions,such as regions 34, 36, each containing bound known-sequence fragments,such as fragment 37 in region 34. The fragments or probes are preferably25-70 bases in lengths and each includes one of the above-identifiedmutations upstream of the CELSR1 gene that is associated with kidneycancer. In particular, the array sequences are designed in length andsequence to bind to those 22q13.3 mutations identified above, but not toassociated fragments. Gene-chip construction, and detection of mutantsequences with such chips, are well known.

In a typical genetic-screening procedure, patient cells are obtained,genomic DNA is extracted, and 22q13.3 sequence regions of interest areamplified by standard PCR, employing fluoresceinated probes. Theamplified material is then reacted with the chip-array sequences, undersuitable hybridization conditions, and the array surface is washed toremove unbound material, then scanned with a suitable chip reader toidentify any mutated sequences associated with kidney cancer. The figureshows binding of a labeled genomic DNA fragment, indicated at 42, to anarray region 38 having bound probe molecules 40. Detection of afluorescent signal in this array region is diagnostic of a known geneticmutation in the critical upstream CELSR1 region of 22q13.3 interval andmay be diagnostic of a genetic predisposition to kidney cancer.

In an alternative embodiment, the mutations identified as above are usedto construct a set of molecular inversion probes (MIPs) capable ofidentifying the presence of genomic mutations. The construction and useof MIPs for identifying genetic mutations have been described (see, forexample, Wang, et al., Nucleic acids research (England) 2005, Vol 33, p.21.

H. Treatment Methods and Pharmaceutical Preparations

The invention also includes methods for treating, e.g., reducing thetumor burden in a human subject with kidney cancer. In one generalimmunotherapy approach, a patient diagnosed with kidney cancer is firstconfirmed as having reduced levels of CELSR1, according to assay methodsdescribed above. If the subject tests positive in this assay, he istreated by administration of anti-CELSR1 antibody. Preferably theantibody is a human or humanized antibody, prepared as described above,and is administered by IV or subcutaneous injection in a suitablephysiological carrier. The antibody dose is preferably 1 to 10 mginjection, and the patient is treated at intervals of every 14 days orso. During treatment, the patient is monitored for change in status ofthe cancer, typically by a combination of a tumor-visualizationprocedure and levels of kidney-related antigens, as above. The treatmentmay be carried out in combination with other kidney-cancer treatments,including drug or radio-isotope therapy, and may be continued until adesired diminution in tumor size is observed.

While the invention has been described with respect to particularembodiments and applications, it will be appreciated that variouschanges and modification may be made without departing from theinvention as claimed.

1. An antibody specific against an epitope contained within amino acidresidues 90-150 of human CELSR1 as defined by SEQ ID NO:1.
 2. Theantibody of claim 1, labeled with a detectable marker suitable forimmunohistochemical detection of cancerous kidney or neuronal tissue,based on the relative absence of histochemical staining of the tissuecompared with normal kidney or neuronal issue, respectively.
 3. Adiagnostic device for use in for screening for kidney cancer in a humansubject, or staging treatment of kidney cancer in a subject, comprising(a) structure for receiving a body-fluid sample from the subject, (b) anantibody specific against a selected domain or epitope of CELSR1, andassociated with said structure and capable of reacting with body-fluidreceived in said structure, to produce, in combination with otherreagents associated with the structure, a detectable reaction indicativeof the presence of CELSR1 sample protein containing that epitope ordomain, and (c) a known-standard indicator against which the level ofdetectable reaction produced can be assessed as a reduced levelassociated with kidney cancer.
 4. The diagnostic device of claim 3,wherein said structure includes a porous pad having the antibodyembedded therein, for reaction with the fluid sample when the sample isapplied to the pad, said detectable reaction is indicated by acalorimetric or fluorimetric indicator, and said known standardindicator includes an indicia that represents a level of CELSR1containing the epitope or domain corresponding to that associated withkidney cancer.
 5. The diagnostic device of claim 3, which includes aspectrophotometric detector for generating a signal related to the levelof CELSR1 produced, a microprocessor for comparing said signal with aknow-standard signal value associated with kidney cancer, and a displayfor displaying an output of the microprocessor.
 6. The diagnostic deviceof claim 3, wherein the anti-CELSR1 binding protein is specific againstan epitope contained within SEQ ID NO:1.
 7. A method for identifyinggenetic mutations associated with an increased risk of kidney cancer,comprising (a) extracting genomic DNA from cells from cancerous kidneytissue from human patients, (b) for the DNA extracted from cells fromeach tissue, comparing the sequence of the DNA in a region selected fromat least one of (i) a plurality of exons 1 to 35 of the CELSR1 onchromosome interval 22q13.3, including adjacent splice site acceptor anddonor sequences of the exons, (ii) a 5′ UTR region within 10 kB or lessof exon 1 of the gene, and (iii) a 3′-UTR region within 10 kB or less ofexon 35, with a homologous region of DNA from cells from normal,wildtype human kidney tissue, (c) by said comparing, identifying one ormore mutations in said regions associated with an increased risk kidneycancer.
 8. The method of claim 7, wherein the DNA that is compared is a5′ UTR region within 10 kB of less of exon 1 of the CELSR gene.
 9. Themethod of claim 8, for use in constructing a gene chip designed forgenetic screening for risk of kidney cancer, further comprising for eachmutation identified, producing a gene fragment capable bindingselectively to genomic DNA fragments carrying that mutation, but not tocorresponding wildtype DNA fragments, and attaching the gene fragmentsconstructed at known positions on a gene-chip substrate.
 10. A method ofscreening for kidney cancer in a human subject, or staging treatment ofkidney cancer in a subject, comprising reacting a body-fluid sample fromthe subject with an antibody specific against a selected domain orepitope of CELSR1, determining from the presence and/or amount ofimmunoassay product, whether the subject has a reduced level of CELSR1protein lacking the specific domain or epitope, when compared with anormal range of CELSR1 in human samples, as an indicator of kidneycancer.
 11. The method of claim 10, wherein the body-fluid sample isurine, and the assayed level of CELSR1 indicative or kidney cancer isless than about 0.1 ng/ml.
 12. The method of claim 10, wherein saidassaying includes applying the body fluid to a solid-phase immunoassaydevice, the level of CELSR1 in the sample is indicated qualitatively bya calorimetric or fluorometric indicator, and said determining includescomparing the indicator with a known standard.
 13. A method of treatingkidney cancer in a subject comprising reacting a body-fluid sample fromthe subject with an antibody specific against a selected domain orepitope of CELSR1, determining from the presence and/or amount ofimmunoassay product, whether the subject has a reduced level of CELSR1protein lacking the specific domain or epitope, when compared with anormal range of CELSR1 in human samples, as an indicator of kidneycancer, and if the subject has such a reduced CELSR1 level,administering a therapeutically effective amount of a CELSR1 antibodyeffective, when bound to the surface of kidney cancer cells, to inhibitgrowth or viability of the cells.
 14. The method of claim 13, whereinthe CELSR1 antibody is human or humanized anti-CELSR1 antibody specificagainst an epitope contained within SEQ ID NO:1.